Concentrated therapeutic phospholipid compositions

ABSTRACT

The invention relates to concentrated therapeutic phospholipid compositions; methods for treating or preventing diseases associated with cardiovascular disease, metabolic syndrome, inflammation and diseases associated therewith, neurodevelopmental diseases, and neurodegenerative diseases, comprising administering an effective amount of a concentrated therapeutic phospholipid composition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/258,044, filed on Sep. 7, 2016, which is a continuation of U.S.application Ser. No. 14/054,588, filed Oct. 15, 2013, which is acontinuation of U.S. application Ser. No. 12/915,724, filed Oct. 29,2010, which claims the benefit of Provisional Application Ser. No.61/256,106, filed Oct. 29, 2009, the contents of all of which areincorporated by reference in their entirety. All patents, patentapplications, and publications cited herein are incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

The invention relates to concentrated therapeutic compositions. Moreparticularly, the invention relates to concentrated therapeuticphospholipid compositions useful for treating or preventing diseases.

BACKGROUND OF THE INVENTION

Genetic traits, coupled with a Western diet and lifestyle, have madecardiometabolic disorders/metabolic syndrome (MetS) a growing globalepidemic. Cardiometabolic syndrome refers to a cluster of cardiovascularrisk factors that include central obesity, high blood pressure, impairedglucose tolerance, hyperglycemia and dyslipidemia. Dyslipidemia is amajor modifiable risk factor leading to atherosclerotic and relatedcardiovascular diseases (CVD) the nation's number one killer.

Cardiovascular Disease

Cardiovascular disease affects one in three people in the United Statesduring their lifetime, and accounts for nearly a third of the deathsthat occur each year (Rosamond W, et al., Circulation, 115, e69-e171,(2007). Cardiovascular diseases are defined as diseases which affect theheart or blood vessels

Statins are considered as first-line therapy for subjects at risk forCVD focusing predominantly on the reduction in low-density lipoproteincholesterol (LDL-C or “bad cholesterol”, to recommended target levels.However statins have minimal effect in raising high-density lipoproteincholesterol (HDL C or “good cholesterol”), now recognized as a majorrisk factor for developing cardiovascular disease. Treatment options toraise HDL-C are very limited and include Niaspan® (branded niacin) whichis known to cause flushing and is reported to cause hepatic enzymeabnormalities, and Tricor® (branded fenofibrates) which causes a 40%increase in LDL C and significant increase in liver enzymes,hematological changes, gall stones, pancreatitis, as well as myopathy.Some treatment options lower plasma triglycerides but have a negligibleeffect on HDL-C (Lovaza®). Other treatment options increase HDL-C, butare less effective on triglycerides.

Others have tried to increase HDL-C (good cholesterol) withoutdeleteriously affecting LDL, TG, or causing hypertension, but have notbeen successful. For example, torcetrapib appeared to raise HDL levels,but had no effect an TGs and LDL. However, torcetrapib caused severehypertension and high mortality in phase III trials. Despiteadvancements in lowering total cholesterol, lipid abnormalities as wellas other severe negative side effects still prevail. Treatment gaps inthe management of dyslipidemia, considered one of the top five majormodifiable risk factors of CVD, represent critical unmet medical needs.While most treatment methods only target the intrinsic LDL-C synthesisin the liver, other treatments are needed to further reducetriglycerides while increasing HDL-C and not increasing LDL-C.

Neurodevelopmental and Neurodegenerative Disease

Neurodevelopmental and neurodegenerative diseases/disorders andneurological imbalance (in neurotransmitters) affect many people, andare defined as chronic progressive neuropathy characterized by selectiveand generally symmetrical loss of neurons in motor, sensory, orcognitive systems. One progressive neurodegenerative disorder,Alzheimer's disease (AD), is irreversible, and is characterized bygradual cognitive deterioration, changes in behavior and personality.These symptoms are related to neurochemical changes, neural death, andthe breakdown of the inter-neural connections. Loss of short-term memoryis often the first sign, followed by cognitive deficits involvingmultiple functions. Early stages of AD and mild cognitive impairment arecharacterized as milder forms of memory loss or cognitive impairmentthat could precede the unset of dementia and AD. Prevention of furthercognitive decline in subjects with these possible precursor conditionsis of paramount importance given that reversibility of AD is notpossible.

It is estimated there are currently about 5.1 million people withAlzheimer's disease (AD) in the United States (Alzheimer's Association,2007) and this number is expected to reach 13.2 million by 2050 (Hebertet al., 2003 Alzheimer's is ranked as the 7th leading cause of death inthe US for people of all ages and the 5th for people aged 65 or older(National Center for Health Statistics, 2004). In Canada it is 280,000people over 65 that are estimated to have AD, and over 750,000 areexpected to have the disease by 2031. (Alzheimer Society of Canada,2006). It is estimated to 10% of all North Americans over the age of 70years nave early stage AD or mild cognitive impairment.

Alzheimer's disease is characterized by two main pathological featuresof the brain: intracellular neurofibrillary tangles formed by abnormalprotein τ (tau); and extracellular neuritic plaques formed by β-amyloidpeptides (Aβ) (Kuo et al., 1996). The overproduction of Aβ42 isgenetically induced but environmental risk factors are required to getfully symptomatic AD (Grant et al., 2002). Among these risk factors, lowdocosahexaenoic acid (DHA) is one of the most important dietary riskfactor for AD (Morris et al., 2005). The reasons for the impact of DHAon learning and memory and the association with AD are unclear but couldresult from its loss in synapses (Montine et al., 2004), which arenormally rich in DHA (Salem et al., 2001), where it is particularlyimportant for postsynaptic transmission and neuroprotection (Bazan,2003). Studies in animal models have consistently showed that brain n-3fatty acid content is highly dependent on dietary intake and aging(Favrere et al., 2000; Youdim et al., 2000; Calon & Cole, 2007).However, some reports claim higher concentrations of DHA have adeleterious effect in neurological patients.

Omega-3 Fatty Acids and Inflammation

Several animal studies, has shown that increased DHA intake has beenfound to increase hippocampal acetycholine levels and its derivatives,neuroprotection DI, which decreased cell death (Aid et al, 2005; Lukiwet al., 2005). A study conducted on aged mice showed that DHA intakeimproved memory performance (Lim et al. 2001). In another Alzheimer'sdisease mouse model, reduction in dietary DHA showed loss ofpostsynaptic proteins associated with increased oxidation, which waslocalized in the dendrites. However, when a group of DHA-restricted micewhere given DHA, they showed signs that the DHA intake protected themagainst dendritic pathology, implying that DHA could be useful inpreventing cognitive impairment in Alzheimers Disease (Calon et al.,2004).

Several epidemiological studies have shown a protective effectassociated with increased fish intake (a direct source of omega 3 fattyacids) against dementia and cognitive impairment decline (Kalmijin etal. 1997, Barberger-Gateau et al. 2002; Morris et al 2003). Recently,one large randomized double-blind placebo-controlled study found 1.6 gDHA and 0.7 EPA may be beneficial in reducing risk for AD (Freund-Leviet al, 2006). In addition, there is mounting evidence that dietarysupplementation with Omega 3 fatty acids may be beneficial in differentpsychiatric conditions such as mood behaviour, depression and dementia(Bourre et al., 2005; Peet and Stokes, 2005; Stoll et al., 1999).

The anti-inflammatory effects of omega-3 fatty acids have been widelystudied with positive results for several chronic inflammatory diseases.C-reactive protein (CRP) is a protein that increases dramatically duringinflammatory processes and is commonly measured as a marker ofinflammation. Greater intake of omega-3 polyunsaturated fatty acid isrelated to a lower prevalence of elevated CRP levels. Animal models ofcolitis indicate that fish oil, a natural source of omega 3 fatty acids,decreases colonic damage and inflammation. Fish oil supplements insubjects with IBD have shown to modulate levels of inflammatorymediators and may be beneficial for the induction and maintenance ofremission in ulcerative colitis. In the management of RA and otherinflammatory conditions, side effects limit the use of NSAIDs, such assalicylates, ibuprofen and naproxen. A clinical trial showed that 39percent of subjects with RA supplemented with cod liver oil were able toreduce their daily NSAID requirement by greater than 30 percent. Omega-3fatty acids have been used to reduce the risk for sudden death caused bycardiac arrhythmias.

Furthermore, omega-3 fatty acid have been shown to improve insulinsensitivity and glucose tolerance in normoglycemic men and in obeseindividuals. Omega-3 fatty acids have also been shown to improve insulinresistance in obese and non-obese subjects with an inflammatoryphenotype. Lipid, glucose and insulin metabolism have been show to beimproved in overweight hypertensive subjects through treatment withomega-3 fatty acids.

Omega-3 fatty acids can be obtained from marine organisms such as squid,fish, krill, etc. and are sold as dietary supplements. However, theuptake of omega-3 fatty acids by the body is not efficient and these rawoils contain other substances such a triglycerides and cholesterol whichare known to cause deleterious side effects such as an increase inLDL-C. Certain fish oils have been developed as pharmaceutical-gradeOM3-acid ethyl esters. One such OM3-acid ethyl ester is presently soldunder the brand name Lovaza®. Studies have shown that Lovaza® candecrease plasma triglycerides levels in patients, however, Lovaza® has anegligible effect on raising good cholesterol (HDL-C). AMR101 is anotherethyl ester form of OM3 fatty acids based on EPA with little or no DHAthat is presently in clinical trials. AMR101 also appears to decreasetriclycerides but also has a negligible effect on raising HDL-C.

A phospholipid composition of OM3 fatty acids as been disclosed in US2004/0234587. This phospholipid composition has OM3 fatty acidsesterified to the phospholipid. This phospholipid composition isreported to be at a concentration of about 40% phospholipids (w/wcomposition) and contains high concentrations of triglycerides (about45%) and free fatty acids (about 15%). When tested in subjects, thiscomposition demonstrated very little effect on lowering triglycerideplasma levels (less than 11% reduction).

Marie oil compositions comprising free fatty acids and lipids, includingOM3 fatty acids and phospholipids, have been disclosed in WO 2000/23546,however the compositions do not disclose OM3 fatty acids esterified todiglycerol phosphate and have very high concentrations of triglyceridesand free fatty acids, and for these reasons would not be expected toreduce triglycerides even to the level of the composition disclosed inUS 2004/0234587, described above.

Therefore, new forms of omega-3 fatty acids are needed that are usefulfor treating or preventing disease. Described herein are novelconcentrated therapeutic phospholipid compositions, as well aspharmaceutical compositions comprising same, and methods of their use.

SUMMARY OF THE INVENTION

Accordingly, in one aspect concentrated therapeutic phospholipidcompositions are described, the compositions comprising compounds of theFormula I:

wherein for each compound of Formula I in the compositions

each R₁ is independently selected from hydrogen or any fatty acid;

each R₂ is independently selected from hydrogen or any fatty acid;

wherein at least one of R₁ and R₂ in each compound of Formula I is afatty acid; and

each X is independently selected from —CH₂CH₂NH₃, —CH₂CH₂N(CH₃)₃ or

wherein the total amount of the compounds of Formula I in thecomposition being at a concentration of between 45% (w/w) to about 99%(w/w).

In some embodiments, the compounds of Formula I the concentratedtherapeutic phospholipid composition are in a concentration of betweenabout 45% (w/w (phospholipids/total composition)) up to 70% (w/w(phospholipids/total composition)). In still further embodiments, thecompounds of Formula I in the concentrated therapeutic phospholipidcomposition are in a concentration of between about 50% (w/w(phospholipids/total composition)) up to 70% (w/w (phospholipids/totalcomposition)). In other embodiments, the compounds of Formula I in theconcentrated therapeutic phospholipid composition are in a concentrationof between about 60% (w/w (phospholipid/total composition)) up to 70%(w/wt (phospholipids/total composition)). In still other embodiments,the compounds of Formula I in the concentrated therapeutic phospholipidcomposition are in a concentration of about 66% (w/w(phospholipids/total composition)).

In other embodiments, the compounds of Formula I in the concentratedtherapeutic phospholipid composition are in a concentration of above 70(w/w (phospholipids/total composition)) to about 99% (w/w(phospholipids/total composition)). In still other embodiments, thecompounds of Formula I in the concentrated therapeutic phospholipidcomposition are in a concentration of between about 85% (w/w(phospholipids/total composition)) to about 95% (w/w(phospholipids/total composition)). In still other embodiments, thecompounds of Formula I in the concentrated therapeutic phospholipidcomposition are in a concentration of between about 85% (w/w(phospholipids/total composition)) to about 95% (w/w(phospholipids/total composition)). In further embodiments, thecompounds of Formula I in the concentrated therapeutic phospholipidcomposition are in a concentration of about 90% (w/w(phospholipids/total composition)).

In some embodiments, R1 is a monounsaturated fatty acid. In otherembodiments, R1 is a polyunsaturated fatty acid. In some embodiments, R2is a monounsaturated fatty acid. In other embodiments. R2 is apolyunsaturated fatty acid. In other embodiments, the polyunsaturatedfatty acid is an omega 3 fatty acid. In still other embodiments, both R1and R2 are each independently selected from an omega 3 fatty acid. Whenat least one of R1 and R2 is an omega 3 fatty acid, the concentratedtherapeutic phospholipid composition comprising compounds of Formula Iis known as an OM3:PL.

In other embodiments, R1 is docosahexaenoic acid (DHA). In otherembodiments, R2 is a monounsaturated fatty acid and R1 is DHA. In otherembodiments, R2 is a polyunsaturated fatty acid and R1 is DHA. In otherembodiments, R2 is an omega 3 fatty acid and R1 is DHA. In still furtherembodiments, R2 is EPA and R1 is DHA. In still further embodiments, R2is DHA and R1 is DHA.

In another embodiments, R1 is eicosapentaenoic acid (EPA). In otherembodiments, R2 is a monounsaturated fatty acid and R1 is EPA. In otherembodiments, R2 is a polyunsaturated fatty acid and R1 is EPA. In otherembodiments, R2 is an omega 3 fatty acid and R1 is EPA. In still furtherembodiments, R2 is DHA and R1 is EPA. In still further embodiments, R2is EPA and R1 is EPA.

In another embodiment, R2 is DHA. In other embodiments, R1 is amonounsaturated fatty acid and R2 is DHA. In other embodiments, R1 is apolyunsaturated fatty acid and R2 is DHA. In other embodiments, R1 is anomega 3 fatty acid and R2 is DHA.

In other embodiments, R2 is EPA. In other embodiments, R1 is amonounsaturated fatty acid and R2 is EPA. In other embodiments, R1 is apolyunsaturated fatty acid and R2 is EPA. In other embodiments, R1 is anomega 3 fatty acid and R2 is EPA.

In some embodiments, the compounds of Formula I in the concentratedtherapeutic phospholipid composition have predominantly DHA at the R2position of Formula I. In other embodiments, there is more DHA in thecompounds of Formula I in the concentrated therapeutic phospholipidcomposition than EPA. In some embodiments, the compounds of Formula I inthe concentrated therapeutic phospholipid composition have greater than60% DHA. In other embodiments, the compounds of Formula I in theconcentrated therapeutic phospholipid composition have greater than 70%DHA. In other embodiments, the compounds of Formula I in theconcentrated therapeutic phospholipid composition have greater than 80%DHA. In other embodiments, the compounds of Formula I in theconcentrated therapeutic phospholipid composition have greater than 90%DHA. In other embodiments, the compounds of Formula I in theconcentrated therapeutic phospholipid composition have greater than 95%DHA.

In some embodiments, there are free fatty acids in the concentratedtherapeutic phospholipid composition, in addition to the fatty acidsesterified to the phosphate. In other embodiments, there are essentiallyno free fatty acids (also expressed as 0% free fatty acids (or FFA)) inthe concentrated therapeutic phospholipid composition.

In other embodiments, the ratio of the total amount of DHA to EPA in theconcentrated therapeutic phospholipid composition is between about 1:1and 1:0.1. In some embodiments the ratio is between about 1:0.7 andabout 1:0.3. In other embodiments, the ratio is about 1:0.5.

In some embodiments, the ratio of the total amount of EPA to DHA in thecompounds of Formula I in the concentrated therapeutic phospholipidcomposition is between about 1:1 and 1:0.1. In some embodiments, theratio is between about 1:0.7 and about 1:0.3. In other embodiments, theratio is about 1:0.5.

In some embodiments, the total amount of OM3 fatty acids in theconcentrated therapeutic phospholipid composition is between about 20%and about 50%. In other embodiments, the total amount of OM3 fatty acidsin the concentrated therapeutic phospholipid composition is betweenabout 30% and about 45%. In other embodiments, the total amount of OM3fatty acids in the concentrated therapeutic phospholipid composition isabout 40%.

In some embodiments, the total, amount of DHA in the concentratedtherapeutic phospholipid composition is between about 5% and 20%. Insome embodiments, the total amount of DHA in the concentratedtherapeutic phospholipid composition is between about 10% and 15%. Insome embodiments, the total amount of DHA in the concentratedtherapeutic phospholipid composition is about 14%.

In some embodiments, the total amount of EPA in the concentratedtherapeutic phospholipid composition is between about 10% and 30%. Insome embodiments, the total amount of DHA in the concentratedtherapeutic phospholipid composition is between about 15% and 25%. Insome embodiments, the total amount of EPA in the concentratedtherapeutic phospholipid composition is about 22%.

In some embodiments, X is —CH₂CH₂NH₃. In other embodiments, X is—CH₂CH₂N(CH₃)₃. In some embodiments, X is

In some embodiments, the concentrated therapeutic phospholipidcomposition comprises predominantly phospholipids containing—CH₂CH₂N(CH₃)₃ (also known as a phosphoyidyl-N-trimethylethanolamine).

In other embodiments, the concentrated therapeutic phospholipidcomposition further comprises an antioxidant. In some embodiments, theantioxidant is a carotenoid. In other embodiments, the carotenoid ispro-vitamin A. In other embodiments, the antioxidant is a flavonoid. Inother embodiments, the flavonoid is selected form naringin, naringenin,hesperetin/kaemferol, rutin, luteolin, neohesperidin, quecertin. Inother embodiments, the flavonoid is

In some embodiments, the concentration of the flavonoid is between about1 mg/kg (w/w of composition) and about 20 mg/kg (w/w of composition). Inother embodiments, the concentration of the flavonoid is greater thanabout 10 mg/kg (w/w of composition).

In further embodiments, the concentrated therapeutic phospholipidcomposition has a concentration of astaxanthin greater than 2000 mg/kg(w/w of composition). In still other embodiments, the concentration ofastaxanthin is between about 2,000 mg/kg (w/w of composition) and about5,500 mg/kg (w/w of composition).

In some embodiments, the concentrated therapeutic phospholipidcompositions has a free fatty acid concentration below about 22% (w/w ofcomposition). In some embodiments, the concentrated therapeuticphospholipid composition has a free fatty acid concentration below about15% (w/w of composition). In some embodiments, the concentratedtherapeutic phospholipid composition has a free fatty acid concentrationbelow about 10% (w/w of composition). In some embodiments, theconcentrated therapeutic phospholipid composition has a free fatty acidconcentration below about 5% (w/w of composition). In some embodiments,the concentrated therapeutic phospholipid composition has a tree fattyacid concentration of about 1% (w/w of composition). In someembodiments, the concentrated therapeutic phospholipid composition has afree fatty acid concentration below 1% (w/w of composition). In someembodiments, the concentrated therapeutic phospholipid composition has afree fatty acid concentration of 0% (w/w of compositions).

In some embodiments, the concentrated therapeutic phospholipidcomposition has a free fatty acid concentration of between about 1%(w/w) and about 20% (w/w). In some embodiments, the concentratedtherapeutic phospholipid composition has a free fatty acid concentrationof between about 5% (w/w) and about 17% (w/w). In some embodiments, theconcentrated therapeutic phospholipid composition has a free fatty acidconcentration of between about 10% (w/w) and about 15% (w/w).

In some embodiments, the concentrated therapeutic phospholipidcomposition has a triglyceride concentration between about 0% (w/w) andabout 30% (w/w). In other embodiments, the concentrated therapeuticphospholipid composition has a triglyceride concentration between about5% and about 20%. In still further embodiments, the concentratedtherapeutic phospholipid composition has a triglyceride concentrationbetween about 101% and about 15%.

In some embodiments, the concentrated therapeutic phospholipidcomposition has a triglyceride concentration below about 15%. In someembodiments, the concentrated therapeutic phospholipid composition has atriglyceride concentration below about 10%. In some embodiments, theconcentrated therapeutic phospholipid composition has a triglycerideconcentration below about 5%. In some embodiments, the concentratedtherapeutic phospholipid composition has a triglyceride concentrationabout 1%. In some embodiments, the concentrated therapeutic phospholipidcomposition has a triglyceride concentration below 1%. In someembodiments, the concentrated therapeutic phospholipid composition has atriglyceride concentration of about 0%.

In other embodiments, the concentrated therapeutic phospholipidcomposition comprises at least 50% compounds of Formula I (w/w), whereinat least 15% of the fatty acid content is EPA, at least 9% of the fattyacid content is DHA, and at least 0.1% astaxanthin (w/w). In otherembodiments, the concentrated therapeutic phospholipid compositioncomprises at least 66% compounds of Formula I (w/w) wherein at least 20%of the fatty acid content is EPA, at least 12% of the fatty acid contentis DHA, and at least 0.4% astaxanthin (w/w). In other embodiments, theconcentrated therapeutic phospholipid composition comprises at least 90%compounds of Formula I (w/w), at least 22% of the fatty acid content isEPA, at least 12% of the fatty acid content is DHA, and 0.4% astaxanthin(w/w).

In other embodiments, the concentrated therapeutic phospholipidcomposition comprises at least 50% compounds of Formula I (w/wcomposition), wherein at least 15% of the fatty acid content is EPA, atleast 9% of the fatty acid content is DHA. In other embodiments, theconcentrated therapeutic phospholipid composition comprises at least 66%compounds of Formula I (w/w), wherein at least 20% of the fatty acidcontent is EPA, at least 12% of the fatty acid content is DHA. In otherembodiments, the concentrated therapeutic phospholipid compositioncomprises above 70% compounds of Formula I (w/w), wherein at least 22%of the fatty acid content is EPA, at least 12% of the fatty acid contentis DHA. In other embodiments, the concentrated therapeutic phospholipidcomposition comprises above 90% compounds of Formula I (w/w), wherein atleast 22% of the fatty acid content is EPA, at least 12% of the fattyacid content is DHA.

In one aspect, a concentrated therapeutic phospholipid composition isdescribed comprising compounds of Formula at a concentration of about66% (w/w (phospholipids/total composition) a free fatty acid (FFA)concentration of less than 6% (w/w FFA/total composition) and atriglyceride concentration of about 0%, the composition being useful fortreating a d preventing cardiometabolic disorders/metabolic syndrome. Insome embodiments, 1 g of the concentrated therapeutic phospholipidcomposition comprises about 387 mg of total OM3 fatty acids wherein EPAis at about 215 mg and DHA is at about 136 mg) and astaxanthin at about5 mg.

In one aspect, a concentrated therapeutic phospholipid composition isdescribed comprising compounds of Formula at a concentration of above70% (w/w (phospholipids/total composition), a free fatty acid (FFA)concentration of about 0% and a triglyceride concentration of about 0%,the composition being useful for treating and preventingneurodegenerative and neurodevelopmental disorders and diseases.

In one aspect the invention is based in part on the unexpected andsurprising discovery that concentrated therapeutic phospholipidcompositions are useful in modulating plasma triglyceride levels as wellas plasma HDL C levels, while not elevating LDL C levels. Thisunexpected ad d surprising discovery is useful in the treatment orprevention of disorders associated with increased triglyceride levels,increased LDL-C levels and decreased HDL-C levels. Such diseases anddisorders include but are not limited to cardiometabolicdisorders/metabolic syndrome (MetS), neurodevelopmental andneurodegenerative diseases/disorders, and inflammation disorders.

In another aspect, a method of treating or preventing a cardiometabolicdisorder/metabolic syndrome is described, the method comprisingadministering to a subject in need thereof a concentrated therapeuticphospholipid composition. In some embodiments, the cardiometabolicdisorder is selected from atherosclerosis, arteriosclerosis coronaryheart (carotid artery) disease (CHD or CAD), acute coronary syndrome (orACS), valvular heart disease, aortic and mitral valve disorders,arrhythmia/atrial fibrillation, cardiomyopathy and heart failure, anginapectoris, acute myocardial infarction (or AMI), hypertension,orthostatic hypotension, shock, embolism (pulmonary and venous),endocarditis, diseases of arteries, the aorta and its branches,disorders of the peripheral vascular system (peripheral arterial diseaseor PAD). Kawasaki disease, congenital heart disease (cardiovasculardefects) and stroke (cerebrovascular disease), dyslipidemia,hypertriglyceridemia, hypertension, heart failure cardiac arrhythmias,low HDL levels, high LDL levels, stable angina, coronary heart disease,acute myocardial infraction, secondary prevention of myocardialinfarction, cardiomyopathy, endocarditis, type 2 diabetes, insulinresistance, impaired glucose tolerance, hypercholesterolemia, stroke,hyperlipidemia, hyperlipoproteinemia, chronic kidney disease,intermittent claudication, hyperphosphatemia, omega-3 deficiency,phospholipid deficiency, carotid atherosclerosis, peripheral arterialdisease, diabetic nephropathy, hypercholesterolemia in HIV infection,acute coronary syndrome (ACS), non-alcoholic fatty liverdisease/non-alcoholic steatohepatitis (NAFLD/NASH), arterial occlusivediseases, cerebral atherosclerosis, arteriosclerosis, cerebrovasculardisorders, myocardial ischemia, coagulopathies leading to thrombusformation in a vessel and diabetic autonomic neuropathy. In someinstances, the methods described above for treating or preventing acardiometabolic disorder/metabolic syndrome may utilize concentratedtherapeutic phospholipid compositions having a concentration of 66% (w/w(phospholipids/composition)).

In another aspect, methods of treating, preventing, or improvingcognition and/or a cognitive disease, disorder or impairment (memory,concentration, learning (deficit)), or of treating or preventingneurodegenerative disorders are described, the method comprisingadministering to a subject in need thereof a concentrated therapeuticphospholipid composition. In some embodiments, the cognitive disease,disorder or impairment is selected from Attention Deficit Disorder(ADD), Attention Deficit Hyperactivity Disorder (ADHD), autism/autismspectrum disorder (ASD), (dyslexia, age-associated memory impairment andlearning disorders, amnesia, mild cognitive impairment, cognitivelyimpaired non-demented, pre-Alzheimer's disease, Alzheimer's disease,epilepsy, Pick's disease, Huntington's disease, Parkinson disease, LouGehrig's disease, pre-dementia syndrome, Lewy body dementia dementia,dentatorubropallidoluysian atrophy, Freidreich's ataxia, multiple systematrophy, types 1, 2, 3, 6, 7 spinocerebellar ataxia, amyotrophic lateralsclerosis, familial spastic paraparesis, spinal muscular atrophy, spinaland bulbar muscular atrophy, age-related cognitive decline, cognitivedeterioration, moderate mental impairment, mental deterioration as aresult of ageing, conditions that influence the intensity of brain wavesand/or brain glucose utilization, stress, anxiety, concentration andattention impairment, mood deterioration, general cognitive and mentalwell being, neurodevelopmental, neurodegenerative disorders, hormonaldisorders, neurological imbalance or any combinations thereof. In aspecific embodiment, the cognitive disorder is memory impairment. Insome instances, the methods described above for treating, preventing, orimproving cognition and/or a cognitive disease, disorder or impairment(memory, concentration, learning (deficit)), or of treating orpreventing neurodegenerative disorders may utilize concentratedtherapeutic phospholipid compositions having a concentration of greaterthan 70% (w/w (phospholipids/composition)).

In another aspect, a method for inhibiting, preventing, or treatinginflammation or an inflammatory disease is described, the methodcomprising administering to a subject in need thereof, a concentratedtherapeutic phospholipid composition. In some embodiments, theinflammation or inflammatory disease is selected from organ transplantrejection; deoxygenation injury resulting from organ transplantation(see Grupp et al., J. Mol. Cell Cardiol. 31: 297-303 (1999)) including,but not limited to, transplantation of the following organs: heart,lung, liver and kidney; chronic inflammatory diseases of the joints,including arthritis, rheumatoid arthritis, osteoarthritis and bonediseases associated with increased bone resorption; inflammatory boweldiseases (IBD) such as ileitis, ulcerative colitis (UC), Barrett'ssyndrome, and Crohn's disease (CD); inflammatory hung diseases such asasthma, acute respiratory distress syndrome (ARDS), and chronicobstructive pulmonary disease (COPD); inflammatory diseases of the eyeincluding corneal dystrophy, trachoma, onchocerciasis, uveitissympathetic ophthalmitis and endophthalmitis; chronic inflammatorydiseases of the gum, including gingivitis and periodontitis;inflammatory diseases of the kidney including uremic complications,glomerulonephritis and nephrosis; inflammatory diseases of the skinincluding sclerodermatitis, psoriasis and eczema; inflammatory diseasesof the central nervous system, including chronic demyelinating diseasesof the nervous system, multiple sclerosis, AIDS-related neurodegeneration and Alzheimer's disease, infectious meningitis,encephalomyelitis, Parkinson's disease, Huntington's disease, Epilepsy,amyotrophic lateral sclerosis and viral or autoimmune encephalitis,preeclampsia; chronic liver failure, brain and spinal cord trauma, andcancer. The inflammatory disease can also be a systemic inflammation ofthe body, exemplified by gram-positive or gram negative shock,hemorrhagic or anaphylactic shock, or shock induced by cancerchemotherapy in response to proinflammatory cytokines, e.g., shockassociated with proinflammatory cytokines. Such shock can be induced,e.g., by a chemotherapeutic agent that is administered as a treatmentfor cancer. Other disorders include depression, obesity, allergicdiseases, acute cardiovascular events, muscle wasting diseases, andcancer cachexia. Also inflammation that results from surgery and traumacan be treated with the concentrated therapeutic phospholipidcompostions.

The details of the invention are set forth in the accompanyingdescription below. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, illustrative methods and materials are now described.Other features, objects, and advantages of the invention will beapparent from the description and from the claims. In the specificationand the appended claims, the singular forms also include the pluralunless the context clearly dictates otherwise. Unless defined otherwise,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. All patents and publications cited in thisspecification are incorporated herein by reference in their entireties.

DESCRIPTION OF THE FIGURES

FIG. 1A depicts a flow chart for the process for making the concentratedtherapeutic phospholipid compositions.

FIG. 1B depicts a flow chart for the process for making the concentratedtherapeutic phospholipid compositions.

FIG. 1C depicts shows the schematic of the supercritical CO2 extractionapparatus.

FIG. 2 depicts circulating plasma triglyceride concentration of C57BL/6mice treated with Composition 3.

FIG. 3 depicts circulating plasma HDL-Cholesterol concentration ofC57BL/6 mice treated with Composition 3.

FIG. 4 depicts circulating plasma percentage of LDL-Cholesterol inC57BL/6 mice treated with Composition 3.

FIG. 5 depicts circulating plasma LDL-Cholesterol concentration ofC57BL/6 mice treated with Composition 3.

FIG. 6 depicts circulating plasma percentage of LDL-Cholesterol inC57BL/6 mice treated with Composition 3.

FIG. 7 depicts circulating plasma NEFA concentration of C57BL/6 micetreated with Composition 3.

FIG. 8 depicts circulating plasma Glucose concentration of C57BL/6 micetreated with Composition 3.

FIG. 9 depicts circulating plasma Phospholipid concentration of C57BL/6mice treated with Composition 3.

FIG. 10 depicts circulating plasma ALT concentration of C57BL/6 micetreated with Composition 3.

FIG. 11 depicts liver Total Cholesterol concentration of C57BL/6 micetreated with Composition 3.

FIG. 12 depicts liver Triglyceride concentration of C57BL/6 mice treatedwith Composition 3.

FIG. 13 depicts circulating plasma triglyceride concentration of LDLr KOmice treated with Composition 3.

FIG. 14 depicts circulating plasma HDL-Cholesterol concentration of LDLrKO mice treated with Composition 3.

FIG. 15 depicts circulating plasma percentage of HDL-Cholesterol in LDLrKO mice treated with Composition 3.

FIG. 16 depicts liver Total Cholesterol concentration of LDLr KO micetreated with Composition 3.

FIG. 17 depicts liver Triglyceride concentration of LDLr KO mice treatedwith Composition 3.

FIG. 18 depicts circulating plasma Triglyceride concentration of ApoA-1CET Tg mice treated with Composition 3.

FIG. 19 depicts circulating plasma total cholesterol concentration ofadult male SD, ZDF, SHR, and JCR:LA rats.

FIG. 20 depicts circulating plasma total cholesterol concentration ofadult male SD, ZDF, SHR and JCR:LA rats.

FIG. 21 depicts circulating plasma HDL/LDL concentration of a adult maleSD, ZDF, SHR and JCR:LA rats.

FIG. 22 depicts circulating plasma total cholesterol/HDL concentrationof adult male SD, ZDF, SHR and JCR:LA rats.

FIG. 23 depicts prothrombin time of adult male SD, ZDF, SHR and JCR:LArats.

FIG. 24 depicts OGTT area under the curve data in ZDF male rats treatedwith Composition 3 for 28 days.

FIG. 25 depicts OGTT area under the curve data in ZDF male rats treatedwith Composition 3 for 28 days.

FIG. 26 depicts OGTT area under the curve data in SD male rats treatedwith Composition 3 for 28 days.

FIG. 27 depicts OGTT area under the curve data in ZDF male rats treatedwith Composition 3 for 28 days.

FIG. 28 depicts the effects of Composition 3 on plasma total cholesterolin male ZDF rats compared to age-matched controls.

FIG. 29 depicts the effects of Composition 3 on plasma HDL-cholesterolin male ZDF rats compared to age-matched controls.

FIG. 30 depicts the effects of Composition 3 on plasma triglycerides inmale ZDF rats compared to age-matched controls.

FIG. 31 depicts the effects of Composition 3 on glucose intolerance inmale ZDF rats.

FIG. 32 depicts the effects of Composition 3 on glucose intolerance inmale ZDF rats.

FIG. 33 depicts the effects of Composition 3 on glucose intolerance inmale SD rats.

FIG. 34 depicts the effects of Composition 3 on glucose intolerance inmale SD rats.

FIG. 35 depicts the comparative effects of Composition 3 and Lovaza® onthe Omega-3 Index.

DETAILED DESCRIPTION OF THE INVENTION

It has been unexpectedly discovered that concentrated therapeuticphospholipid compositions demonstrate surprising effects in thetreatment of metabolic disorders, cardiovascular disease,neurodevelopmental disorders and neurodegenerative diseases, andinflammation disorders.

Definitions

The following definitions are used in connection with the concentratedtherapeutic phospholipid compositions:

The term “concentrated therapeutic phospholipid composition” and“concentrated therapeutic phospholipid compositions” as used hereinrefer to the concentrated therapeutic phospholipid compositionscomprising compounds of Formula I.

The articles “a” and “an” are used in this disclosure to refer to one ormore than one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “and/or” is used in this disclosure to mean either “and” or“or” unless indicated otherwise.

The term “about” when used in this disclosure along with a recited valuemeans the value recited and includes the range of + or −5% of the value.For example, the phrase about 80% means 80% and + or −5% of 80, i.e. 76%to 84%. The recited value “about 0%” as used herein means that thedetectable amount is less than one part per thousand.

The term “fatty acid” or “fatty acid residue” as used herein means acarboxylic acid with a long unbranched aliphatic chain, which is eithersaturated or unsaturated. Saturated fatty acids have the general formulaC_(n)H_(2n)+1 COOH. Examples of saturated fatty acids include but arenot limited to: Propanoic acid, Butanoic acid, Pentanoic acid, Hexanoicacid, Heptanoic acid, Octanoic acid, Nonanoic acid, Decanoic acid,Undecanoic acid, Dodecanoic acid, Tridecanoic acid, Tetradecanoic acid,Pentadecanoic acid, Hexadecanoic acid, Heptadecanoic acid, Octadecanoicacid, Nonadecanoic acid, Eicosanoic acid, Heneicosanoic acid, Docosanoicacid, Tricosanoic acid, Tetracosanoic acid, Pentacosanoic acid,Hexacosanoic acid, Heptacosanoic acid, Octacosanoic acid, Nonacosanoicacid, Triacontanoic acid, Henatriacontanoic acid, Dotriacontanoic acid,Tritriacontanoic acid, Tetratriacontanoic acid, Pentatriacontanoic acid,Hexatriacontanoic acid. An unsaturated fat is a fat or fatty acid inwhich the are one or more double bonds in the fatty acid chain. A fatmolecule is monounsaturated if it contains one double bond, andpolyunsaturated if it contains more than one double bond. Examples ofunsaturated fatty acids include but are not limited to: Myristoleicacid, Palmitoleic acid, Sapience acid, Oleic acid, Linoleic acid,α-Linolenic acid, Arachidonic acid, Eicosapentaenoic acid (EPA), Erucicacid, Docosahexaenoic acid (DHA), and Docosapentaenoic acid.

A “subject” is a mammal, e.g., a human, mouse, rat, guinea pig, dog,cat, horse, cow, pig, or non-human primate, such as a monkey chimpanzee,baboon or rhesus.

Representative “pharmaceutically acceptable salts” include, e.g.,water-soluble and water-insoluble salts, such as the acetate, amsonate(4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzoate,bicarbonate, bisufate, bitartrate, borate, bromide, butyrate, calcium,calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate,dihydrochloride, edetate, edisylate, estolate, esylate, fumarate,gluceptate, gluconate, glutamate, glycollylarsanilate,hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide,hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate,lactobionate, laurate, magnesium, malate, maleate, mandelate, mesylate,methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate,N-methylglucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate,oxalate, palmitate, pamoate (1,1-methene-bis-2-hydroxy-3-naphthoate,einbonate), pantothenate, phosphate/diphosphate, picrate,polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate,subacetate, succinate, sulfate, sulfosalicylate, suramate, tannate,tartrate, teoclate, tosylate, triethiodide, and valerate salts.

The term “metabolic disorder” as used herein refers to disorders,diseases and syndromes involving dyslipidemia, and the terms metabolicdisorder, metabolic disease, and metabolic syndrome are usedinterchangeably herein.

An “effective amount” when used to describe an amount of a concentratedtherapeutic phospholipid composition useful for treating or preventing adisease or disorder, is an amount that is efficacious with respect tothe disease or disorder connected with that particular effective amount.

The term “carrier”, as used in this disclosure, encompasses carriers,excipients, and diluents and means a material, composition or vehicle,such as a liquid or solid filler, diluent, excipient, solvent orencapsulating material, involved in carrying or transporting apharmaceutical agent from one organ, or portion of the body, to anotherorgan, or portion of the body.

The term “treating”, with regard to a subject, refers to improving atleast one symptom of the subjects disorder. Treating can be curing,improving, or at least partially ameliorating the disorder.

The term “disorder” is used in this disclosure to mean, and is usedinterchangeably with, the terms disease, condition, or illness, unlessotherwise indicated.

The term “administer”, “administering”, or “administration” as used inthis disclosure refers to either directly administering a compound orpharmaceutically acceptable salt of the compound or a composition to asubject, or administering a prodrug derivative or analog of the compoundor pharmaceutically acceptable salt of the compound or composition tothe subject, which can form an equivalent amount of active compoundwithin the subjects body.

Methods of Making the Concentrated Therapeutic Phospholipid Composition

The concentrated therapeutic phospholipid compositions can be made orproduced by any method known to one of skill in the art. For example,phospholipid containing oils can be isolated from natural sources (seeUS 2004/0234587, US 2009/0074857, and US 2008/0274203 the disclosures ofwhich are incorporated by reference in their entireties), which can thenbe further processed. Alternatively, following the process outlined inFIG. 1a results in bulk raw material krill oil ready for furtherprocessing. These phospholipid containing oils can be further processedusing countercurrent supercritical CO₂ extraction (Lucien, F. P., etal., Australas Biotechnol. 1993, 3, 143-147) to concentrate thecompositions to produce the concentrated therapeutic phospholipidcompositions described herein (see FIG. 1b ). For example,countercurrent supercritical CO₂ extraction at 70 C and 30 MPa and witha CO₂/oil ratio of 72 can be used to remove certain biomolecules such asall triglycerides from the bulk raw material krill oil as well as someof the free fatty acids (FIG. 1b ). As more of the TGs and FFAs areremoved from the bulk raw material krill oil, the concentration of thephospholipids increases. When the TGs have been removed through thisprocess the phospholipid composition is at about 66% concentration (w/w(phospholipids/composition)) and contains less than 5% free fatty acids(w/w). As more of the FFAs are removed using this process, aconcentrated therapeutic phospholipid composition results having aphospholipid concentration above 70% up to about 90% (w/w(phospholipids/composition)) having about 1% or less TG and about 0%FFA. Other aquatic and/or marine biomasses may be used as startingmaterials, such as, for example, squid or blue mussels. Additionalcomponents can be added before, during, or after processing.Alternatively, phospholipids can be synthesized; a typical way tosynthesize would be, among others, according to the procedure describedin U.S. Pat. No. 7,034,168, the disclosure of which is incorporatedherein its entirety.

Method for Using the Concentrated Therapeutic Phospholipid Compositions

Described herein are methods of reducing circulating plasmaconcentrations of triglycerides, LDL-cholesterol, total cholesterol andNEFA, the method comprising administering to a subject in need thereofan effective amount of a Composition of the Invention.

Also provided are methods of increasing plasma concentrations ofHDL-cholesterol and hepatic concentrations of triglycerides and totalcholesterol, the method comprising administering to a subject in needthereof an effective amount of a concentrated therapeutic phospholipidcomposition.

In another aspect, a method of reducing TG without the risk ofincreasing LDL is descried, the method comprising administering to asubject in need thereof, a concentrated therapeutic phospholipidcomposition.

Also provided are methods for inhibiting, preventing, or treating ametabolic disorder, or symptoms of a metabolic disease, in a subject,the method comprising administering to a subject in need thereof aneffective amount of a concentrated therapeutic phospholipid composition.Examples of such disorders include, but are not limited toatherosclerosis, dyslipidemia, hypertriglyceridemia, hypertension, heartfailure, cardiac arrhythmias, low HDL levels, high LDL levels, stableangina, coronary heart disease, acute myocardial infarction, secondaryprevention of myocardial infarction, cardiomyopathy, endocarditis, type2 diabetes, insulin resistance, impaired glucose tolerance,hypercholesterolemia stroke, hyperlipidemia, hyperlipoproteinemia,chronic kidney disease, intermittent claudication, hyperphosphatemia,carotid atherosclerosis, peripheral arterial disease, diabeticnephropathy, hypercholesterolemia in HIV infections acute coronarysyndrome (ACS), non-alcoholic fatty liver disease, arterial occlusivediseases, cerebral atherosclerosis, arteriosclerosis, cerebrovasculardisorder, myocardial ischemia, and diabetic autonomic neuropathy.

Also provided are methods for inhibiting, preventing, or treatinginflammation or an inflammatory disease in a subject. The inflammationcan be associated with an inflammatory disease. Inflammatory diseasescan arise where there is an inflammation of the body tissue. Theseinclude local inflammatory responses and systemic inflammation, Examplesof such diseases include, but are not limited to: organ transplantrejection; reoxygenation injury resulting from organ transplantation(see Grupp et al., J. Mol Cell Cardiol, 31: 297-303 (1999)) including,but not limited to, transplantation of the following organs: heart,lung, liver and kidney; chronic inflammatory diseases of the joints,including arthritis, rheumatoid arthritis, osteoarthritis and bonediseases associated with increased bone resorption; inflammatory boweldiseases such as ileitis, ulcerative colitis, Barrett's syndrome, andCrohn's disease; inflammatory lung diseases such as asthma, adultrespiratory distress syndrome, and chronic obstructive airway disease;inflammatory diseases of the eye including corneal dystrophy, trachoma,onchocerciasis, uveitis, sympathetic ophthalmitis and endophthalmitis;chronic inflammatory diseases of the gum, including gingivitis andperiodontis; inflammatory diseases of the kidney including uremiccomplications, glomerulonephritis and nephrosis; inflammatory diseasesof the skin including sclerodermatitis, psoriasis and eczema;inflammatory diseases of the central nervous system, including chronicdemyelinating diseases of the nervous system, multiple sclerosis,AIDS-related neurodegeneration and Alzheimer's disease, infectiousmeningitis, encephalomyelitis, Parkinson's disease, Huntington'sdisease, amyotrophic lateral sclerosis and viral or autoimmuneencephalitis. Metabolic disease such as type II diabetes mellitus; theprevention of type I diabetes; dyslipdemia; diabetic complications,including, but not limited to glaucoma, retinopathy, nephropathy, suchas microaluminuria and progressive diabetic nephropathy, polyneuropathy,atherosclerotic coronary arterial disease, peripheral arterial disease,nonketotic hyperglycemichyperosmolar coma, mononeuropathies, autonomicneuropathy, joint problems, and a skin or mucous membrane complication,such as an infection, a shin spot, a candidal infection or necrobiosislipoidica diabeticorum; immune-complex vasculitis, systemic lupuserythematosus; inflammatory diseases of the heart such ascardiomyopathy, ischemic heart disease hypercholesterolemia, andatherosclerosis; as well as various other diseases that can havesignificant inflammatory components, including preeclampsia; chronicliver failure, brain and spinal cord trauma, and cancer. Theinflammatory disease can also be a systemic inflammation of the body,exemplified by gram-positive or gram negative shock, hemorrhagic oranaphylactic shock, or shock induced by cancer chemotherapy in responseto proinflammatory cytokines, e.g., shock associated withproinflammatory cytokines. Such shock can be induced, e.g., by achemotherapeutic agent that is adminstered as a treatment for cancer.Other disorders include depression, obesity, allergic diseases, acutecardiovascular events, muscle wasting diseases, and cancer cachexia.Also inflammation that results from surgery and trauma can be treatedwith the concentrated therapeutic phospholipid compositions.

Also provided are methods for inhibiting, preventing, or treatinghypertriglyceridemia in subject. In some embodiments, thehypertriglyceridemia is moderate hypertriglyceridemia. In someembodiments, the subject is diagnosed with moderatehypertriglyceridemia. Moderate hypertriglyceridemia is defined as asubject having a TG level of >3.9 mmol/L (>350 mg/dL).

Also provided are methods for reducing tasting plasma levels ofLow-density Lipoprotein Cholesterol (LDL-C) in a subject. In someembodiments of reducing fasting plasma levels of Low-density LipoproteinCholesterol (LDL-C), the subject is diagnosed with moderatehypertriglyceridemia.

Also provided are methods for increasing fasting plasma levels ofHigh-density Lipoprotein Cholesterol (HDL-C) in a subject. In someembodiments of increasing fasting plasma levels of High-densityLipoprotein Cholesterol (HDL-C), the subject is diagnosed with moderatehypertriglyceridemia.

Also provided are methods for increasing the Omega-3 index (OM3I) in asubject. The Omega-3 Index is defined as the percentage of EPA+DHA inred blood cells (RBC) which can be represented by the formula:OM3I=(EPA+DHA)/Total fatty acids in RBC. Low levels of EPA+DHA inerythrocytes are associated with increased risk for sudden cardiac deathand can be viewed as a marker of increased risk (an actual risk factor)for death from coronary heart disease (Harris, 2010). In otherembodiments, the method provided elevates the omega-3 index (OM3I) andreduces oral glucose intolerance (OGTT). In some embodiments ofincreasing omega-3 index, the subject is diagnosed with moderatehypertriglyceridemia.

Also provided are methods for reducing high sensitivity C-reactiveprotein (hs-CRP) in a subject. In some embodiments of reducing highsensitivity C-reactive protein (hs-CRP), the subject is diagnosed withmoderate hypertriglyceridemia.

Also provided are methods for inhibiting, preventing, or treatingcardiovascular disease in a subject. Cardiovascular diseases includeatherosclerosis, arteriosclerosis, coronary artery disease, heart valvedisease, arrhythmia, heart failure, hypertension, orthostatichypotension, shock, endocarditis, diseases of the aorta and itsbranches, disorders of the peripheral vascular system, and congenitalheart disease.

Also provided are methods for inhibiting, preventing, or treatingmetabolic syndrome in a subject. Metabolic syndrome is a combination ofmedical disorders that increase the risk of developing cardiovasculardisease and diabetes. It affects one in five people, and prevalenceincreases with age. Some studies estimate prevalence in the USA to be upto 25% of the population. Metabolic syndrome is also known as metabolicsyndrome X, syndrome X, insulin resistance syndrome, Reaven's syndrome,and CHAOS (Australia).

Also provided are methods for inhibiting, preventing, or treating acognitive disorder, in a subject. The term “cognitive disease ordisorder” as used herein should be understood to encompass any cognitivedisease or disorder. Non-limiting examples of such a cognitive diseaseor disorder are Attention Deficit Disorder (ADD), Attention DeficitHyperactivity Disorder (ADHD), dyslexia, age-associated memoryimpairment and learning disorders, amnesia, mild cognitive impairment,cognitively impaired non-demented, pre-Alzheimer's disease, autism,dystonias and Tourette syndrome, dementia, age related cognitivedecline, cognitive deterioration, moderate mental impairment, mentaldeterioration as a result of ageing, conditions that influence theintensity of brain waves and/or brain glucose utilization, stress,anxiety, concentration and attention impairment, mood deterioration,general cognitive and mental well being, neurodegenerative disorders,hormonal disorders or any combinations thereof. In a specificembodiment, the cognitive disorder is memory impairment.

The term “improving a condition in a subject suffering from a cognitivedisease or a cognitive disorder” as used herein should be understood toencompass: ameliorating undesired symptoms associated with a disease,disorder, or pathological condition; preventing manifestation ofsymptoms before they occur; slowing down progression of a disease ordisorder; slowing down deterioration of a disease or disorder; slowingdown irreversible damage caused in a progressive (or chronic) stage of adisease or disorder; delay ng onset of a (progressive) disease ordisorder; reducing severity of a disease or disorder; curing a diseaseor disorder; preventing a disease or disorder from occurring altogether(for example in an individual generally prone to the disease) or acombination of any of the above. For example, in a subject sufferingfrom memory impairment, for example as a result of Alzheimer's Disease,symptoms including deterioration of spatial short-term memory, memoryrecall and/or memory recognition are improved by use of a lipidconcentrated therapeutic phospholipid composition.

Also provided are methods for inhibiting, preventing, or treatingneurodegenerative disorder in a subject. Neurodegenerative disorder isdefined as a chronic progressive neuropathy characterized by selectiveand generally symmetrical loss of neurons in motor, sensory, orcognitive systems. Non limiting examples of neurodegenerative disordersinclude but are not limited to Alzheimer's disease, Pick's disease, Lewybody dementia Basal ganglia-Huntington's disease, Parkinson's disease,dentatorubropallidoluysian atrophy, Fredreich's ataxia, multiple systematrophy, types 1, 2, 3, 6, 7 spinocerebellar ataxia Motor-amyotropiclateral sclerosis, familial spastic paraparesis, spinal muscularatrophy, spinal and bulbar muscular atrophy, Lou Gehrig's disease,pre-dementia syndrome, Lewy body dementia, age-related cognitivedecline, cognitive deterioration, moderate mental impairment, mentaldeterioration as a result of ageing, dentatorubropallidoluysian atrophy,Freidreich's ataxia, multiple system atrophy, types 1, 2, 3, 6, 7spinocerebellar ataxia, amyotrophic lateral sclerosis, and familialspastic paraparesis.

Also provided are methods for reducing the decline of global cognitivefunction in a subject. In some embodiments, the reduction in decline ofglobal cognitive function can be measured by the Neuropsychological TestBattery (NTB). In some embodiments, the subject is diagnosed with earlystage Alzheimer's disease.

Also provided are methods for reducing worsening of neuropsychiatricsymptoms in a subject. In some embodiments, the reduction is measured bythe Neuropsychiatric Inventory questionnaire (NPI). In some embodimentsthe subject is diagnosed with early stage Alzheimer's disease.

Also provided are methods for maintaining self-care and activities ofdaily living function in a subject suffering from Alzheimer's disease.In some embodiments, the subject is diagnosed with early stageAlzheimer's disease. In some embodiments, the maintaining self-care andactivities of daily living function is measured by the DisabilityAssessment in Dementia caregiver-based interview (DAD).

Additional health disorders or conditions which may be treated orimproved by the concentrated therapeutic phospholipid compositionsinclude, but are not limited to, high blood cholesterol levels, hightriglycerides levels, high blood fibrinogen levels, low HDL/LDL ratio,menopausal or post-menopausal conditions, hormone related disorders,vision disorders, immune disorders, liver diseases, chronic hepatitis,steatosis, lipid peroxidation, dysrhythmias of cell regeneration,destabilization of cell membranes, high blood pressure, cancer,hypertension, aging, kidney disease, skin diseases, edema,gastrointestinal diseases, peripheral vascular system diseases,allergies, airways diseases, and psychiatric diseases.

In some embodiments, the subject is administered an effective amount ofa concentrated therapeutic phospholipid composition. In otherembodiments, the treatment comprises a combination of a concentratedtherapeutic phospholipid composition and treatment agents such asanti-dyslipidemic agents. Anti-dyslipidemic ages include but are notlimited to atorvastatin, fluvastatin, lovastatin, pitavastatin,pravastatin, rosuvastatin and simvastatin.

In other embodiments, the treatment comprises a combination of aconcentrated therapeutic phospholipid composition and a cholinesteraseinhibitor. Cholinesterase inhibitors include but are not limited tometrifonate (irreversible), carbamates, physostigmine, neostigmine,pyridostigmine, ambenonium, demarcarium, rivastigmine, phenanthrenederivatives, galantamine, piperidines, donepezil, tacrine, edrophonium,huperzine A, ladostigill and ungeremine.

In some embodiments, the subject is administered a combination of aconcentrated therapeutic phospholipid composition and at least one ofvitamins, minerals, cox-inhibitors, sterols, fibrates,antihypertensives, insulin, cholesterol digestion inhibitors, forexample ezetimibe, fatty acids, omega-3 fatty acids, antioxidants, andthe methylphenidate class of compounds, such as for example ritalin. Inother embodiments, a combination of a concentrated therapeuticphospholipid composition and elements depleted during traditionalchronic treatments, such as for example during chronic treatment withstatins. For example, in some embodiments, a concentrated therapeuticphospholipid composition is described which contains at least one ofcox-2, folic acid, vitamin B6, vitamin B12, magnesium or zinc. In otherembodiments, combination therapies comprising a concentrated therapeuticphospholipid composition and potassium are described. Potassium isusually depleted during treatment with diuretics. Combination therapiesreduce risk of side effects, increase benefits, increase solubility,and/or increase bioavailability.

Modes of Administration

Administration of the concentrated therapeutic phospholipid compositionscan be accomplished via any mode of administration for therapeuticagents. These modes include systemic or local administration such asoral, parenteral transdermal, subcutaneous, or topical administrationmodes.

Pharmaceutical Formulations

Depending on the intended mode of administration, the compositions canbe in solid, semi-solid or liquid dosage form, such as, for example,injectables, tablets, pills, time-release capsules, elixirs, tinctures,emulsions, syrups, liquids, suspensions, or the like, some time in unitdosages and consistent with conventional pharmaceutical practices.Likewise, they can also be administered in intravenous (both bolus andinfusion) intraperitoneal, subcutaneous or intramuscular form, all usingforms well known to those skilled in the pharmaceutical arts.

Illustrative pharmaceutical compositions are tablets and gelatincapsules comprising a concentrated therapeutic phospholipid compositionneat, or if required, contains a pharmaceutically acceptable carrier,such as a) a diluent, e.g., purified water, triglyceride oils, such ashydrogenated or partially hydrogenated vegetable oil, or mixturesthereof, corn oil, olive oil, sunflower oil, safflower oil, lactose,dextrose, sucrose, mannitol, sorbitol, cellulose, sodium, saccharin,glucose and/or glycine; b) a lubricant, e.g., silica, talcum, stearicacid, its magnesium or calcium salt, sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chlorideand/or polyethylene glycol; for tablets also; c) a binder, e.g.,magnesium aluminum silicate, starch paste, gelatin, tragacanth,methylcelluose, sodium carboxymethylcellulose, magnesium carbonate,natural sugars such as glucose or beta-lactose, corn sweeteners, naturaland synthetic gums such as acacia, tragacanth or sodium alginate, waxesand/or polyvinylpyrrolidone, if desired; d) a disintegrant, e.g.,starches, agar, methyl cellulose, bentonite, xanthan gum, algic acid orits sodium salt, or effervescent mixtures; e) absorbent, colorant,flavorant and sweetener; f) an emulsifier or dispensing agent, such asTween 80, Labrasol, HPMC, DOSS, caproyl 909, labrafac, labrafil, peceol,transcutol, capmul MCM, capmul PG-12, captex 355, gelucire, vitamin ETGPS or other acceptable emulsifier; and/or g) an agent that enhancesabsorption of the compound such as cyclodextrin,hydroxypropyl-cyclodextrin, PEG400, PEG200.

Liquid, particularly injectable, compositions can, for example, beprepared by dissolution, dispersion, etc. For example, the concentratedtherapeutic phospholipid composition is dissolved, in or mixed with apharmaceutically acceptable solvent such as, for example, water, saline,aqueous dextrose, glycerol, ethanol, and the like, to thereby form aninjectable isotonic solution or suspension. Proteins such as albumin,chylomicron particles, or serum proteins can be used to solubilize theconcentrated therapeutic phospholipid composition.

Other illustrative topical preparations include creams, ointments,lotions, aerosol sprays and gels, wherein the concentration of theconcentrated therapeutic phospholipid composition ranges from about 0.1%to about 15%, w/w or w/v.

Dosing

The dosage regimen utilizing the concentrated therapeutic phospholipidcompositions is selected in accordance with a variety of factorsincluding type, species, age, weight, sex and medical condition of thesubject; the severity of the condition to be treated; the route ofadministration; the renal or hepatic function of the subject; and theparticular concentrated therapeutic phospholipid composition employed. Aphysician or veterinarian of ordinary skill in the art can readilydetermine and prescribe the effective amount of the drug required toprevent, counter or arrest the progress of the condition.

Effective dosage amounts of the present invention, when used for theindicated effects, range from about 20 mg to about 10000 mg of theconcentrated therapeutic phospholipid composition per day, Dosages forin vivo or in vitro use can contain about 20, 50, 75, 100, 150, 250,500, 750, 1000, 1250, 2500, 3500, 5000, 7500 or 10000 mg of theconcentrated therapeutic phospholipid composition. Effective bloodplasma levels after administration of the concentrated therapeuticphospholipid composition to a subject can range from about 0.002 mg toabout 100 mg per kg of body weight per day. Appropriate dosages of theconcentrated therapeutic phospholipid composition can be determined asset forth in L. S. Goodman, et al., The Pharmacological Basis ofTherapeutics, 201-26 (5th ed. 1975).

The concentrated therapeutic phospholipid compositions can beadministered in a single daily dose, or the total daily dosage can beadministered in divided doses of two, three or four times daily. To beadministered in the form of a transdermal delivery system, the dosageadministration can be continuous rather than intermittent throughout thedosage regimen. In some embodiments, of combination therapy, theconcentrated therapeutic phospholipid composition and the therapeuticagent can be administered simultaneously. In other embodiments, theconcentrated therapeutic phospholipid composition and the therapeuticagent can be administered sequentially. In still other embodiments ofcombination therapy, the concentrated therapeutic phospholipidcomposition can be administered daily and the therapeutic agent can beadministered less than daily. In still other embodiments of combinationtherapy, the concentrated therapeutic phospholipid composition can beadministered daily and the therapeutic agent can be administered morethan once daily.

EXAMPLES

The disclosure is further illustrated by the following examples, whichare not to be construed as limiting this disclosure in scope or spiritto the specific procedures herein described. It is to be understood thatthe examples are provided to illustrate certain embodiments and that nolimitation to the scope of the disclosure is intended thereby, it is tobe further understood that resort may be had to various otherembodiments, modifications, and equivalents thereof which may suggestthemselves to those skilled in the art without departing from the spiritof the present disclosure and/or scope of the appended claims.

Concentrated Therapeutic Phospholipid Compositions

The following non-limiting examples of therapeutic compositions serve toillustrate further embodiments of the concentrated therapeuticphospholipid composition. It is to be understood that any embodimentslisted in the Examples section are embodiments of the concentratedtherapeutic phospholipid composition and, as such, are suitable for usein the methods and compositions described above.

The following methods can be used to make the concentrated therapeuticphospholipid compositions (FIGS. 1A and 1B)

Step 1:

Frozen krill is mechanically crushed and incubated with a solvent in aratio of 9:1 acetone water for 60-90 minutes at 8° C. to extractdifferent proportions of the lipids (PL, TG and FFA) from the krillbiomass. Lipids are subsequently separated from proteins and krillmaterial by filtration under pressure (50-60 kpa). The solid phase isdiscarded. The soluble extract is evaporated by a continuousdistillation column under vacuum to remove the solvent (acetone). Themajor part of the aqueous (water) fraction is separated from the lipidfraction by decantation and the remaining water removed by evaporationunder vacuum and gentle heating. Those fractions are dosed, analyzed,and blended to constitute an intermediary krill oil product which isre-analyzed to achieve desired specifications ±5%:EPA (15 g/100 g), DHA(9 g/10 g), total phospholipids (42 g/100 g) and astaxanthin's forms(125 mg/100 g).

Step 2:

100.5 g of received krill oil from step 1 was charged to a 300 mlextraction vessel (ID=0.68″). The extractor was sealed, pre-heated CO2at 55° C. was introduced from the bottom, and the pressure in theextractor was maintained at 5,000 psi using a diaphragm CO2 pump. Theflow of CO2 was continued in the upflow direction through the extractorand was expanded to atmospheric pressure through apressure-reduction-valve (PRV) so that the dissolved material in the CO2precipitated and collected in the flask. The flow rate and volume of CO2exiting the flask was measured with a flowmeter and dry test meter(DTM). A total of 7200 g of CO2 was passed through the extractor(solvent to feed ratio, S/F=72) and 34.1% of the charge was removed bythe CO2. The low of about 25 Standard Liters/min of CO2 was maintainedduring the course of the test and the total time of extraction was about160 min. The extractor was isolated and the CO2 was vented toatmosphere. The extractor was opened and the un-extracted material(raffinate-product) was removed from the vessel.

Step 3:

SC CO2 Extraction to produce 90+% OM3:PLs. 9.44 g of oil was mixed withinert packing and charged to the extractor. The procedure carried outsimilar to that described in step 2 except moor aggressive extractionconditions were used with the pressure and temperature in the extractormaintained at 10,000 psi and 70° C. A total S/F ratio of 200 was used f;therefore, about 1900 g of CO2 was flowed through the extractor. A flowrate of CO2 of about 10 Standard Liters/min was maintained; therefore,the total run time for this test was 105 min. A total of 56.3% of thecharge was extracted from this oil during the course of the run. Theextractor was isolated, the CO2 vented to atmosphere, the vessel opened,and the resulting raffinate-product scraped off the inert packing. Thisun-extracted material analyzed to be 91%, OM3:PLs.

Composition 1

47% OM3:PL *Total lipids as FA_(TG) g/100 g oil 61.3 (100)   *Omega-3g/100 g oil 14.1 (22.5) *EPA g/100 g oil 7.4 (11.6) *DHA g/100 g oil 3.8 (6.1) *DPA g/100 g oil 0.2  (0.3) *Omega-6 g/100 g oil 10.8 (18.3)*Linoleic acid g/100 g oil 10.6 (18.0) *Omega-9 g/100 g oil 6.6 (11.6)*Oleic acid g/100 g oil 6.1 (10.8) *Sat. FA_(TG) g/100 g oil 21.4 (36.1)*Monounsat. FA_(TG) g/100 g oil 13.9 (23.1) *Polyunsat._(TG) g/100 g oil26.0 (40.7) *EPA as FA_(TG) g/100 g oil 7.7 *DHA as FA_(TG) g/100 g oil3.9 Water % 0.8 Color — Red orange Odor — Slightly rancid Totalcarotenoids mg/100 g oil 36.0 Astaxanthine mg/100 g oil 65.3Astaxanthine % diester 83.1 % monoester 16.9 % free 0.0 Peroxide IndexmEq peroxide/kg 1.0 p-Anisidine Index — 2.0 Iodine Index gI₂/100 g oil101.1 Saponification Index mg KOH/g oil 214.1 Indice acide mg KOH/g oil17.2 Total fat % Free fatty acid % as oleic acid 5.2 Triglycerides %36.5 Viscosity cP 1323.0 ash % 5.0 Vitamin A UI/g Oil 40.4 Vitamin EUI/g Oil 0.1 Total phospholipids g/100 g oil 47.2 Phospholipid profileTLC — % LPC 3.7 % PC 53.6 % PS 24.7 % PE 16.4 % PA 1.7 Molecular mass PLg/mol 773.8

Composition 2

53% OM3:PL Total lipids as fatty acids (FA) TG g/100 g oil 69.80 TotalOmega-3 g/100 g oil 31.30 C 20:5 (n = 3) EPA g/100 g oil 13.90 C 22:6 (n= 3) DHA g/100 g oil 10.10 C 22:5 (n = 3) DPA g/100 g oil 0.40 TotalOmega-6 g/100 g oil 1.60 linoleic acid - LA g/100 g oil 1.30 TotalOmega-9 g/100 g oil 6.10 oleic acid - OA g/100 g oil 5.70 Saturated FAg/100 g oil 21.10 Monounsaturated FA g/100 g oil 14.50 PolyunsaturatedFA g/100 g oil 34.20 EPA as FA g/100 g oil 14.40 DHA as FA g/100 g oil10.50 PHOSPHOLIPID PROFILE total g/100 g oil 52.30 lysophosphatidylcholine - LPC % 10.80 sphingomyeline - SM % 0.10 phsophatidyl choline -PC % 79.70 phsophatidyl serine - PS % phsophatidyl inositol - PI %phosphatidyl ethanolamine - PE % 9.40 PA % 0.00 CAROTENOIDS total mg/100g oil 92.60 total astaxanthin - AST mg/100 g oil 161.60 AST diester62.00 AST monoester 35.00 AST free 3.00

Composition 3

66% OM3:PL Total lipids as fatty acids (FA) TG g/100 g oil 74.2 TotalOmega-3 g/100 g oil 39.8 C 20:5 (n = 3) EPA g/100 g oil 21.7 C 22:6 (n =3) DHA g/100 g oil 14.1 C 22:5 (n = 3) DPA g/100 g oil 0.5 Total Omega-6g/100 g oil 1.7 linoleic acid - LA g/100 g oil 1.3 Total Omega-9 g/100 goil 5.8 oleic acid - OA g/100 g oil 5.1 Saturated FA g/100 g oil 18.0Monounsaturated FA g/100 g oil 13.2 Polyunsaturated FA g/100 g oil 43.1EPA as FA g/100 g oil 22.6 DHA as FA g/100 g oil 14.6 PHOSPHOLIPIDPROFILE total g/100 g oil 66.2 lysophosphatidyl choline - LPC % 10.7phsophatidyl choline - PC % 75.3 phosphatidyl ethanolamine - PE % 11.8other % 2.2 CAROTENOIDS total mg/100 g oil 273.4 total astaxanthin - ASTmg/100 g oil 466.8 AST diester % 57.4 AST monoester % 40.7 AST free %1.9

Composition 4

80% OM3:PL Total lipids as fatty acids (FA) TG g/100 g oil 68.35 TotalOmega-3 g/100 g oil 37.90 C 20:5 (n = 3) EPA g/100 g oil 20.40 C 22:6 (n= 3) DHA g/100 g oil 12.95 C 22:5 (n = 3) DPA g/100 g oil 0.48 TotalOmega-6 g/100 g oil 1.45 linoleic acid - LA g/100 g oil 1.26 TotalOmega-9 g/100 g oil 4.93 oleic acid - OA g/100 g oil 4.35 Saturated FAg/100 g oil 16.15 Monounsaturated FA g/100 g oil 11.21 PolyunsaturatedFA g/100 g oil 40.99 EPA as FA g/100 g oil 21.30 DHA as FA g/100 g oil13.50 PHOSPHOLIPID PROFILE total g/100 g oil 80.00 lysophosphatidylcholine - LPC % 9.20 sphingomyeline - SM % 0.20 phsophatidyl choline -PC % 80.60 phsophatidyl serine - PS % 1.10 phsophatidyl inositol - PI %0.10 phosphatidyl ethanolamine - PE % 7.50 PA % 1.30 CAROTENOIDS totalmg/100 g oil 180.4 total astaxanthin - AST mg/100 g oil 325.5 ASTdiester % 68.45 AST monoester % 29.27 AST free % 2.28

Composition 5

90% OM3:PL *Total lipids as FA_(TG) g/100 g oil 63.9 *Omega-3 g/100 goil 35.1 *EPA g/100 g oil 18.9 *DHA g/100 g oil 12.2 *DPA g/100 g oil0.5 *Omega-6 g/100 g oil 1.3 *Linoleic acid g/100 g oil 1.2 *Omega-9g/100 g oil 4.6 *Oleic acid g/100 g oil 3.9 *Sat. FA_(TG) g/100 g oil15.8 *Monounsat. FA_(TG) g/100 g oil 10.2 *Polyunsat._(TG) g/100 g oil37.9 *EPA as FA_(TG) g/100 g oil 19.7 *DHA as FA_(TG) g/100 g oil 12.7Acetone Ppm 1.6 Humidity and volatiles % 1.7 Water % 1.9 Color — Redchili Odor — shellfish Total carotenoids mg/100 g oil 168.9 Astaxanthinemg/100 g oil 309.3 Astaxanthine % Diester 73.1 % Monoester 25.3 % Libre1.6 Index p-Anisidine — 3.1 Index acid mg KOH/g oil 33.6 Index iodinegI₂/100 g oil Index saponification mg KOH/g oil Index Peroxide mEqperoxyde/kg 0.1 Vitamin A UI/g Oil 15.2 Vitamin E UI/g Oil 0.3 Fattyacid total % 97.6 Viscosity cP Total phospholipids g/100 g oil 90.6Phospholipid profile TLC — % LPC 13.5 % SM 0.4 % PC 76.3 % Autres PL 1.2% PE 7.9 % PA 0.8 Triglycerides % 0.0

Composition 6

70% OM3:PL derived from Squid *Total lipids as FA_(TG) g/100 g oil 54.5*Omega-3 g/100 g oil 29.1 *EPA g/100 g oil 8.9 *DHA g/100 g oil 18.3*DPA g/100 g oil 0.2 *Omega-6 g/100 g oil 0.7 *Linoleic acid g/100 g oil0.3 *Omega-9 g/100 g oil 4.2 *Oleic acid g/100 g oil 2.0 *Sat. FA_(TG)g/100 g oil 16.9 *Monounsat. FA_(TG) g/100 g oil 6.8 *Polyunsat._(TG)g/100 g oil 30.9 *EPA as FA_(TG) g/100 g oil 9.3 *DHA as FA_(TG) g/100 goil 19.1 Humidity (calmar) % Indice acide mg KOH/g oil 55.7 Vitamin AUI/g Oil Vitamin E UI/g Oil Fatty acid total % 2.7 Total carotenoidsmg/100 g oil 8.3 Astaxanthine mg/100 g oil 13.2 Astaxanthine % diester42.5 % monoester 35.6 % libre 21.9 Total phospholipids g/100 g oil 70.8*Phospholipid profile TLC — % LPC 12.4 % SM 7.8 % PC 55.8 % other 2.0 %PE 22.0 % PA 6.4 Triglycerides % 25.0 Free fatty acid % as oleic acid3.2 Index p-Anisidine — 4.3 Index Peroxide mEq peroxyde/kg 0.6 **PMPhospholipids (g/mol) 847.14 Profile of Fatty Acids of the PL *Totallipids as FA g/100 g PL 53.9 *Omega-3 g/100 g PL 28.6 *EPA g/100 g PL9.0 *DHA g/100 g PL 18.3 *DPA g/100 g PL 0.2 *Omega-6 g/100 g PL 0.5*Linoleic acid g/100 g PL 0.2 *Omega-9 g/100 g PL 3.6 *Oleic acid g/100g PL 1.5 *Sat. FA g/100 g PL 18.3 *Monounsat. FA g/100 g PL 5.4*Polyunsat. FA g/100 g PL 30.2

Biological Examples Example 1

Managing Dyslipidemia in Three Murine Phenotypes

The aim of this study was to examine the effects of Composition 3 inthree age-/sex-matched murine phenotypes representative of (1) normalhealthy non-obese normoglycemic control (C57BL6) versus (2)hyperdislipidemic LDL-receptor gene knockout (LDLr −/−) or (3) humanapoA-I transgenic mice (Jackson Labs) at 12-w of age: 27.5±0.7 vs25.6±0.7 vs 29.2±0.8 gr, respectively; n=7-10/gr, kept according tolocal and national ethic regulations, fed a normal vs a Western-dietregime and water ad libitum. Data are presented as mean±sem andstatistics assessed by t-test (unpaid, two-tail) (v5-GraphPad Prism).

The profile of plasma lipids concentrations (mg/dL) in the above-threeuntreated adult male murine models were as reported in the literature:Total cholesterol (TC): 71.1±3.3 vs 215.3±10.4 vs 50.3±1.3;triglycerides (TGs): 59.5±4.5 vs 65.1±3.8 vs 53.0±12.9; low-densitylipoprotein (LDL): 13.3±1.2 vs 101.6±6.7 vs 12.2±1.6; high-densitylipoprotein (HDL): 53.4±3.2 vs 88.8±3.6 vs 24.8±2.6. Six (6) weeks of QDtreatment with Compression 3 (104 vs 208 vs 417 mg/kg (Human equivalentdosing of 500, 1,000 and 2,000 mg/day) in C57BL6 led to significantdose-dependent decrease in plasma TGs (up to 60%), % reduced LDL (up to28%) elevated HDL (by 17%) but did not affect TC (see FIGS. 2-12 andTable 1). In severely dyslipidemic LDLr-KO mice, Composition 3 led tosignificant dose-dependent decrease in plasma TGs, elevated further HDL,caused a slight elevation in TC (only at mid-dose) and did not affectLDL (see FIGS. 13-17 and Table 1). In hApoA-I transgenic mice,Composition 3 led to significant decrease in plasma TGs, elevated HDLand did not affect TC (see FIG. 18 and Table 1). The liverconcentrations of TC were the same in all three phenotypes but TGs werereduced (19%; p<0.05) in LDLr-KO and elevated (153%; p<0.01) in hApoA-Icompared to control C57BL6. Treatment with Composition 3 elevated liverTC and TGs by up to 12% and 27%, respectively, in C57BL6, by up to 10%and 36% in LDLr-KO and by up to 10% for TC but mixed effects for TGs(−13 to +12%) in hApoA-I mice, respectively (see Table 2).

TABLE 1 Effects mediated by 6-weeks treatment with Composition 3 onplasma lipids. Low dose Mid-dose High-dose Murine Plasma 104 mg/kg 208mg/kg 417 mg/kg phenotype Lipids (HED: 500 mg/day) (HED: 1000 mg/day)(HED: 2000 mg/day) C57BL6 TC 75.9 ± 2.0 80.6 ± 3.3 78.1 ± 2.7 up 6.8%(NS) up 13.4% (NS) up 9.8% (NS) TGs 32.2 ± 1.6 26.0 ± 2.8 23.8 ± 1.2down 46% (p < 0.001) down 56% (p < 0.001) down 60% (p < 0.001) LDL 11.1± 1.4 11.2 ± 1.0  9.6 ± 0.7 down 15% (NS) down 16% (p ≤ 0.05) down 28%(p < 0.05) HDL 59.4 ± 1.9 63.4 ± 3.5 62.3 ± 2.4 up 11% (NS) up 19% (NS)up 17% (p < 0.05) LDLr-KO TC 219.3 ± 7.4  244.4 ± 7.9  238.5 ± 6.9  up2% (NS) up 14% (p < 0.01) up 11% (NS) TGs 45.7 ± 2.8 41.7 ± 4.9 36.7 ±1.6 down 30% (p < 0.001) down 36% (p < 0.01) down 44% (p < 0.001) LDL99.2 ± 5.7 116.2 ± 4.4  97.4 ± 6.5 down 2% (NS) up 14% (NS) down 4% (NS)HDL 90.9 ± 2.5 88.4 ± 4.6 111.2 ± 2.5  up 2% (NS) =(NS) up 25% (p <0.001) hApoA-I TC 51.1 ± 1.5 57.2 ± 2.6 50.1 ± 1.4 transgenic up 2% (NS)up 14% (p < 0.05) =(NS) TGs 19.3 ± 2.8  43.2 ± 11.7 43.0 ± 8.2 down 64%(p < 0.05) down 18% (NS) down 19% (NS) LDL 10.8 ± 1.0 12.8 ± 2.3 13.9 ±1.0 down 11% (NS) =(NS) up 14% (NS) HDL 27.9 ± 2.4 28.6 ± 2.6  21.6 ±12.8 up 13% (NS) up 15% (NS) down 13% (NS) NS; not significant

TABLE 2 Plasma lipids at baseline between murine phenotype C57BL6Variation vs hApoA-I Variation control LDLr-KO control transgenic vscontrol Plasma concentrations (mg/dL) total cholesterol (TC) 71.1 ± 3.3215.3 ± 10.4 up 3-fold 50.3 ± 1.3 down 29.3% p < 0.001 NS triglycerides(TGs) 59.5 ± 4.5 65.1 ± 3.8 up 9.4%  53.0 ± 12.9 down 10.9% NS NS lowdensity lipoprotein 13.3 ± 1.2 101.6 ± 6.7  up 7.6-fold 12.2 ± 1.6 down8.3% (LDL-C) p < 0.001 NS high-density lipoprotein 53.4 ± 3.2 88.8 ± 3.6up 166% 24.8 ± 2.6 down 54% (HDL-C) p < 0.001 p < 0.001 Liverconcentrations (μg/mg) total cholesterol (TC) 23.1 ± 0.8 23.6 ± 0.6=(NS) 25.1 ± 0.5 up 9% (NS) triglycerides (TGs) 53.0 ± 3.1 42.9 ± 2.1down 19% 81.3 ± 7.4 up 153% p < 0.05 p < 0.001 NS; not significant

These data indicate that the Composition 3 is an effective modulator oflipid metabolism, mainly at reducing plasma TGs and LDL and elevatingHDL. These data indicate that in some embodiments, concentratedtherapeutic phospholipid compositions can be effective as a therapyagainst moderate to severe hypertriglyceridemia. In some embodiments,concentrated therapeutic phospholipid compositions in combination withother anti-dyslipidemic agents can be effective at lowering refractoryhypertriglyceridemia.

Example 2

Increase of the Circulating Plasma Concentration of High-DensityLipoprotein-Cholesterol (HDL-C) and Reduced Total Cholesterol (TC)/HDLRatio in 12-Week Old Male Zucker Diabetic Fatty Rats.

The purpose of this study was to examine the effects of the concentratedtherapeutic phospholipid compositions in the Zucker Diabetic Fatty ratrodent model for type 2 diabetes with obesity, hyperlipidaemia andinsulin, resistance (ZDF; Gmi-fa/fa) vs age-/sex-matched normal healthynon-obese normoglycemic lean control SD rat (from Charles River Labs;12-w, 359±17 vs 439±13 gr; n=9-12/gr.). Lipid profile (total cholesterol(TC), triglycerides (TGs), High-Density Lipoprotein Cholesterol (HDL-C)and TC/HDL ratio were assessed before, 1 and 2 month after QD treatmentwith Composition 3 (52 versus 260 mg/kg (HED of 500 and 2,500 mg), andkept according to local and national ethic regulations (Formulab highfat 5008 (ZDF) vs normal 5001 (SD) diet regime and water ad libitum).Data are presented as Mean±SD (n=2-10) and statistical differencescalculated by unpaired two-tailed t test (v5-GraphPad Prism). At 12-w ofage, the circulating plasma concentrations of TC, TGs, HDL and TC/HDLratio were: 4.6±0.9, 11.6±5.9, 2.3±1.1 mmol/L and 2.16±0.62,respectively. Lipids' profile in SD rats were significantly lower at1.9±0.4, 1.2±0.4, 1.3±0.2 mmol/L and 1.45±0.11, respectively. Daily lowand high dose treatment for 60 days did not affect TC and TGsconcentrations but increased by 1.7- to 1.8-fold (p<0.01) “good”HDL-cholesterol and decreased the TC/HDL ratio by 26-32% (p<0.01-0.05),respectively.

Example 3

Improved Glucose Intolerance in Zucker Diabetic Fatty Rats afterAdministration of Composition 3

The purpose of this study was to investigate the effects of Composition3 in an overly dislipidemic, obese, type 2 diabetic rat model. ZuckerDiabetic Fatty (ZDF; Gmi-fa/fa) rats were used versus age-/sex-matchednormal healthy non-obese normoglycemic lean control SD rat (from CharlesRiver Labs; 12-w, 359±17 vs. 439±13 gr; n=9-12/gr.). Glucose intolerancewas assessed conducting an oral glucose tolerance test (OGTT; overnightfasting then single gavage of glucose 2 g/kg rat b.w.) over 180 minutesusing glucometer strips (Accu-Chek Aviva, Roche Diagnostics), before and90 days after treatment with Composition 3 given by QD gavage at 52versus 260 mg/kg (HED of 500 and 2,500 mg), and kept according to localand national ethic regulations (Formulab high fat 5008 (ZDF) vs normal5001 (SD) diet regime and water ad libitum). Data are presented asMean±SD and statistical differences calculated by unpaired two-tailed ttest (v5-GraphPad Prism).

At 12-w of age (T₀) fasting circulating plasma concentrations of glucosewere 7.8≅2.1 vs 5.0±0.6 mmol/L (p<0.001) in untreated ZDF vs SD rats.Non-fasting ZDF and SD rats glucose levels were 22.0±4.2 vs 8.6±0.6mmol/L, respectively. One month later, baseline values increased by1.9-fold (p<0.0001) in fasted ZDF while remaining unchanged in fastedSD. Aging did not affect glucose levels in non-fasted rats. Glucosechallenge led to a maximum 2.5-fold (p<0.0001) and 1.6-fold (p<0.0001)increase in plasma glucose concentrations in untreated fasted ZDF andSD, at 30 and 60 minutes, respectively, returning mostly to initialvalues after 180 mins. At 16 weeks of age, thirty days (T₃₀) oftreatment did not affect either the profile (AUC) or maximum elevationin glucose in SD rats but treatment of ZDF shifted to the right themaximum elevation in plasma glucose (from 30 to 60 minuets), reduced by61-72% (p<0.02) the peak elevation at 30 minutes and reduced by 50-60%(p<0.0001) the AUC at either doses of Composition 3, thus back to theAUC observed in untreated glucose challenged SD rats. At 20 weeks ofage, 60 days of treatment, either dosing did not further attenuateglucose intolerance. None of the treatment profile affected the plasmaand urinary concentrations of glucose (hyperglycemia and glucosuria) innon-fasted ZDF or SD. These data indicate that a short term and low dosechronic administration of Composition 3 significantly improves glycemiccontrol in a model of severe hyperglycemia.

Example 4

A Randomized, Placebo-Controlled, Double-Blind, Dose-Ranging andMulti-Centered Trial to Evaluate the Safety and Efficacy of ConcentratedTherapeutic Phospholipid Compositions in the Treatment of ModerateHypertriglyceridemia.

Subjects with moderate hypertriglyceridemia treated by physicianaccording to the Canadian Lipid Treatment Guidelines who are treatedover 12 weeks with concentrated phospholipid given at doses of 1.0, 2.0or 4.0 g. The primary measure of efficacy will be the percent change infasting blood circulating serum triglycerides (TGs) between baseline(Week 1) and 12 weeks of treatment. Secondary Outcomes: between baselineand after six weeks and 12 weeks of treatment: 1) absolute change infasting plasma TGs; 2) percentage (%) of subjects achieving target TGfasting plasma levels; 3) absolute change in fasting plasma LDL-C,VLDL-C, HDL-C, HDL-C, HDL2-C, HDL3-C, Total Cholesterol, hs-CRP andnon-HDL; 4) percentage (%) change in fasting plasma concentrations ofLDL-C, VLDL-C, HDL-C, HDL2-C, HDL3-C, TC, hs-CRP and non-HDL; 5)calculated Ratios: a) total cholesterol: HDL-C; b) LDL-C; HDL-C; c) TGs:HDL-C; 6) LDL-C-related parameters: a) particle number; b) particlesize; c) oxidation; 7) absolute and percent (%) change in fasting plasmaconcentrations of biomarkers; a) glycated Hemoglobin (HbA1c), b)apolipoprotein A-I (ApoA-I), c) apolipoprotein B-100 (ApoB-100), d)apolipoprotein E (ApoE), e) lipoprotein(a) (Lp(a)), f) adiponectin, g)glucose, h) insulin; 8) calculated ApoB:ApoA-I ratio; 9) fasting plasmalipoprotein-associated phospholipase A2 activity (Lp-PLA2); 10) HOMA-IR(homeostasis model assessment of insulin resistance: [glucose(mmol/L)×IRI (microIU/L)/22.5]; 11) plasma concentrations of total EPAand DHA (PK/PD—25 subjects/group); 12) OM3I (Omega-3 index); 13;Subjects Genetic Polymorphism: a) Lecithin:Cholesterol Acyltransferase(LCAT), b) cholesteryl ester transfer protein (CETP), e) scavengerreceptor type B-1 (SR-B1), d) ATP Binding Cassette transporter 1(ABCA1).

Example 5

Preclinical Non-GLP Assessment of Efficacy of Concentrated TherapeuticPhospholipid Compositions Alone or in Combination with a Statin onModulating Blood Lipids and the Development of Atherosclerotic Lesionsin ApoE Null Mice Fed a Western-Type Diet.

Male adult mice (n=135 (15 mice/group) 5-6 weeks of age) weighing about18-20 g each homozygous for the Apoetm 1 Unc mutation are administeredHOW either Vehicle (Water or 0.2%-0.5% t carboxymethylcellulose);composition 3 (1,000 mg/daily HED); composition 3 (2,000 mg/daily HED);or Lipitor (20 mg/daily HED); or composition 3 (1,000 mg/dailyHED)+Lipitor (20 mg/daily HED). At 0, 3 months, or 6 months thefollowing assessment of values is made relative to: blood Lipids: TC,TGs, LDL, HDL, non-HDL, VLDL, (0, 3 and 6 months) (2) AorticAtherosclerosis (0, 3 and 6 months): a. Thoracic and abdominal aortawill be isolated, trimmed of fit, laid out and pinned on black matrixfor photography, and stained with Sudan IV or Oil Red-O. b. Vessel willbe imaged for surface involvement using a computerized image analysissystem (image ProPlus or NIH Package Software). The data will becomputed by group and statistically analyzed. c. Lipid extraction:Following staining and morphometric analysis, aortas will be extracted(Bligh/Dyer). (3) Red blood cells Omega-3 Index (0, 3 and 6 months); (4)Circulating plasma concentration of CRP (0, 3 and 6 months).

Example 6 Comparison of Composition 3 with Lovaza® on the Omega 3 Index

Male adult (14 weeks) Sprague-Dawley (SD) rats with an average bodyweight of >375-425 were fed normal rat chow (diet 5075-normal, standardrat chow). Number of test subjects/group: n=56; n=8 rats/gr. Dosing wasQD (single daily dosing/morning) for 12 weeks with either (i) Vehicle(ii) Composition 3 52 mpk 500 mg/day HED; (iii) Composition 3 104 mpk1000 mg/day HED; (iv) Composition 3 416 mpk 4000 mg/day HED; (v) Lovaza®416 mpk=4000 mg/day HED. Results are shown in FIG. 35.

Example 7

Monotherapy Study of Concentrated Phospholipids in Early StageAlzheimer's Disease

Subjects will be randomly assigned to receive either concentratedtherapeutic phospholipid composition 1 g, fish oil (135 mg EPA: 108 mgDHA) 1 g, or placebo (soy oil) 1 g once daily. The primary outcomemeasure will be the change in NTB between baseline and 24 weeks oftreatment. The Neuropsychological Test Battery (NTB) will be used tomonitor and evaluate important cognitive changes. The following 9components of the NTB are used to determine the outcome for the subject:(1) Wechsler Memory Scale, visual immediate (score range, 0-18), (2)Wechsler Memory Scale verbal immediate (score range, 0-24), (3) ReyAuditory Verbal Learning, Test (RAVLT) immediate (score range, 0-105),(4) Wechsler, Memory Digit Span (score range, 0-24), (5) Controlled WordAssociation, Test (COWAT), (6) Category Fluency Test (CT), (7) Wechsler,Memory Scale visual delayed (score range, 0-6), (8) Wechsler, MemoryScale verbal delayed (score range, 0-8), and (9) RAVLT, delayed (scorerange, 0-30)(Harrison et al. 2007). The RAVLT delayed measure iscomposed of delayed recall and recognition performance components thatare summed to yield a score ranging from 0 to 30, yielding 9 measures ofsubject performance. Secondary outcome measures will include the changein the NPI and DAD at 24 weeks of treatment. The NPI evaluates 12neuropsychiatric disturbances common in dementia: delusions,hallucinations, agitation, dysphoria anxiety, apathy, irritability,euphoria, disinhibition, aberrant motor behaviour, night-time behaviourdisturbances, and appetite and eating abnormalities. The DAD is acaregiver-based interview instrument used to evaluate instrumental andbasic activities of daily living in dementia (hygiene, dressing,undressing, continence, eating, meal preparation telephoning, going onan outing, finance, correspondence, medication, leisure and housework).The NPI also assesses the amount of caregiver distress engendered byeach of the neuropsychiatric disorders. Blood is drawn and levels ofEPA, DHA and phospholipids is measured.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

The invention claimed is:
 1. A pharmaceutical composition comprisingcompounds of the Formula I:

wherein R1 and R2 each independently represent a docosahexaenoic acid(DHA) or an eicosapentaenoic acid (EPA) residue; each X is independentlyselected from —CH₂CH₂NH₃, —CH₂CH₂N(CH₃)₃,

and free EPA and free DHA, wherein the total free and bound EPA is at aconcentration of between 15% and 25% (w/w), and the total free and boundDHA is at a concentration of between 10% and 15% (w/w), and a carrier;wherein the total amount of the phospholipid compounds of Formula I inthe composition are at a concentration of about 50% (w/w) to about 70%(w/w).
 2. The composition of claim 1, wherein the phospholipid compoundsof Formula I are at a concentration of about 55% (w/w).
 3. Thecomposition of claim 1, wherein the phospholipid compounds of Formula Iare at a concentration of about 50-60% (w/w).
 4. The composition ofclaim 1, further comprising an antioxidant.
 5. The composition of claim4, wherein the antioxidant is selected from astaxanthin, a carotenoid,vitamin A and vitamin E.
 6. The composition of claim 5, wherein theantioxidant is vitamin E.
 7. The composition of claim 5, wherein theantioxidant is astaxanthin.
 8. The composition of claim 1, furthercomprising triglycerides in a concentration of less than 5% by weight.9. The composition of claim 1, said composition formulated as a capsule.10. The composition of claim 1, wherein the phospholipid compounds ofFormula I are at a concentration of 55% (w/w) ±5%.
 11. The compositionof claim 1, wherein said composition is formulated as a capsule or apill.
 12. A method of reducing triglyceride level in a patient,comprising administering to said patient the composition of claim
 1. 13.The method of claim 12, wherein the level of high-density lipoprotein(HDL) in said patient are further increased.
 14. The method of claim 12,wherein the level of low-density lipoprotein (LDL) in said patient arefurther reduced.
 15. The method of claim 12, wherein said patient hashypertriglyceridemia.